PROJECT SUMMARY/ABSTRACT Approximately 90% of glioblastoma patients develop another tumor in two years after surgical removal and the treatment of radiation and temozolomide (TMZ), indicating that recurrent glioblastoma cells become resistant to TMZ. This resistance significantly lowers the clinical efficacy of TMZ, thereby contributing to the high incidence of tumor recurrence. One well known mechanism of TMZ resistance is related to the function of O-6- methylguanine-DNA methyltransterase (MGMT); this enzyme repairs TMZ-induced DNA damage, allowing cancer cells to replicate and spread. However, targeting MGMT has limited clinical benefits. We have recently demonstrated that a PI3K (phosphatidylinositol-4,5-biphosphate 3-kinase) catalytic subunit is a specific biomarker for the prognosis of glioblastoma recurrence. Class IA PI3K family includes PIK3CA, B, and G catalytic subunits, encoding p110?, ?, and ?, respectively. Targeting all these subunits to restore TMZ sensitivity has been widely studied; however, this treatment is often associated with non-selective killing and severe toxicity. Uncovering the selective role of PI3K subunits in TMZ resistance represents an alternative mechanism that is independent of MGMT and offers a new approach to enhancing TMZ treatment efficacy and reducing the risk of tumor recurrence. We hypothesize that (1) PIK3CB/p110? is critical for the survival of TMZ- resistant cells and (2) p110? inhibitors restore TMZ sensitivity of glioblastoma cells and recurrent tumors, while exhibiting limited toxicities to normal brain cells. We will test these hypotheses via the following specific aims: (1) To define the selective role of p110? in TMZ resistance (Year 1) and (2) To determine the efficacy of p110? inhibitors in circumventing TMZ resistance and treating tumor recurrence in mice (Years 1-2). To test the first hypothesis, we will measure the effect of four PI3K catalytic subunits (PIK3CA, B, D, and G) in the survival of p110?-high/TMZ-resistant glioblastoma cells, glioblastoma stem cells, human astrocytes, or neural stem cells. We will test whether p110? but not other PI3K subunits will induce TMZ resistance and determine whether other exogenous PI3K subunits will substitute for endogenous p110? to induce TMZ resistance. To test the second hypothesis, we will measure the therapeutic effect of PI3K isoform-selective inhibitors in TMZ- resistant GBM cells/GSCs in vitro and in vivo. Elucidating the selective role of PIK3CB/p110? in TMZ resistance and glioblastoma recurrence will explain the selective roles of PI3K genes in glioblastoma and will further foster the rational design of novel therapies tailored to glioblastoma patients with an especially high risk of tumor recurrence.